Cyclic AMP is a major second messenger that converts an extracellular signal to an intracellular signal. The enzyme responsible for its formation is adenylyl cyclase (AC), which is a membrane bound enzyme that catalyzes the conversion of ATP to cyclic AMP (cAMP) upon stimulation of various Gs protein-coupled receptors by neurotransmitters and hormones. Cyclic AMP then activates protein kinase A (PKA) by dissociating its regulatory subunit from the catalytic subunit (Scott, J. D, (1991) Pharmac. Ther. 50:123-145). The catalytic subunit of PKA then initiates an enzymatic cascade of phosphorylation reactions within the cell, for example, various enzymes involved in myocyte contraction in the heart, such as troponin (Holroyde, M. J., Robertson, S. P., Johnson, J. D., Solaro, R. J., and Potter, J. D (1980), J. Biol. Chem. 255:11668-11693), phospholamban (Kim, H. W., Steenaart, N. A. E., Ferguson, D. G., and Kranias, E. G, (1990), J. Biol. Chem. 265:1702-1709), those involved in glucose metabolism in the liver, such as glycogen phosphorylase (Krebs, E. G. (1985), Biochem. Soc. Trans. 13:813-820), and those involved in neuronal function (Nishi, A., Bibb, J. A., Snyder, G. L., Higashi, H., Naim, A. C., and Greengard, P. (2000), Proc. Natl. Acad. Sci. U S A 97:12840-12845). By phosphorylating uniquely differentiated proteins in each cell type, cAMP signaling and thus PKA regulate the unique function of each organ. Cyclic AMP is eventually degraded to AMP by phosphodiesterase (Soderling, S. H., and Beavo, J. A. (2000) Curr Opin Cell Biol 12:174-179). PKA is inactivated by re-association of the catalytic subunit with the regulatory subunit (Ishikawa, Y. (1998), Regulation ofcAMP signaling by phosphorylation. In Adenylyl cyclases. D. M. F. Cooper, editor. Philadelphia: Lippincott-Raven Publishers. 99-120). Phosphorylated proteins are dephosphorylated by phosphatases, thereby pulling the protein conformation back into an inactive form.
Molecular cloning studies have elucidated the presence of at least 9 isoforms of adenylyl cyclase that differ in biochemical properties and tissue distribution. All AC isoforms share the same membrane topology, i.e., a tandem repetition of a six-transmembrane domain and a large cytoplasmic domain. The amino acid sequence within the membrane domain is not conserved among these isoforms; however, that of the cytoplasmic domain is relatively well-conserved and is considered to be the catalytic domain. Interestingly, a group of isoforms shows higher amino acid sequence homology with each other than with other isoforms. Subsequent biochemical studies also revealed that certain isoforms share not only a similar amino acid sequence, but also display similar biochemical properties. Based upon amino acid sequence homology, biochemical properties, and tissue distribution, the nine isoforms can be subdivided into at least five subgroups. Importantly, the diversity in their biochemical properties, in particular, regulation by calmodulin and G beta/gamma subunits, and in their tissue distribution may explain the conflicting findings of earlier studies in which membrane preparations from a variety of different tissues were used for AC assays. Furthermore, the finding of diversity in the AC isoforms and in their regulation during the past decade has expanded our understanding of this classic intracellular signaling pathway. One of the questions yet remaining is the specific role of each isoform in a given cell type and organ function. A particular AC isoform must play a specific role in regulating the physiological function of a given organ. Unfortunately, previous in vitro experimental approaches were unable to address this issue.
The distribution of the AC isoforms within the brain is heterogeneous, suggesting that each isoform is involved in a distinct aspect of neuronal signaling (Cooper, D., editor. (1998), Adenylyl cyclases. Philadelphia: Lippincott-Raven Publishers). The hippocampus is rich in AC1 and since this isoform is activated by Ca-calmodulin, it has been speculated that it plays a role in long-term potentiation mediated by the glutamate receptor. The olfactory bulb is rich in AC3. AC5 is most dominant in the striatum, implicating its involvement in motor regulation (Glatt, C. E., and Snyder, S. H. (1993) Nature 361:536-538). AC5 is located mostly in medium-sized striatal neuronal cells expressing D1 dopaminergic receptors in the basal ganglia, and accordingly has been implicated in signal detection to dopaminergic function. In contrast, most AC6 is present in most neurons and is co-localized with various neurotransmitters systems, AC6 might be in regulation of the classical neuronal signal integration on the brain (Liu, F. C., Wu, G. C., Hsieh, S. T., Lai, H. L., Wang, H. F., Wang, T. W., and Chem, Y. (1998), FEBS Lett 436:92-98). However, the role of these isoforms in neuronal function in vivo is poorly understood. A key question that remains unanswered is what the specific role of an AC isoform is in neuronal function and cAMP signal whose expression is limited to a specific brain region.
A number of neurotransmitters and neuromodulators in the brain are mediated though G protein-coupled receptors, including those of the classical neurotransmitters, dopamine, serotonin, and adrenaline. All the AC isoforms are subject to the regulation of G proteins and thus AC is a crucial molecule in modulating the physiological responses of this broadly expressed neurotransmission and neuromodulation system. The diversity of the AC family members may allow each isoform to function in a different signal transduction pathway of neurotransmitters, neuromodulators or neurotrophic factors. This is particularly important for the neuronal system, unlike the heart, in which a single neuron may receive stimulating and/or sequential multiple inputs from other neurons in a fraction of a second. Further, the mode of this input may differ from one region of the brain to another. The coincidence detector of AC renders neurons capable of detecting simultaneous stimulation of two or more neurotransmitters. This neuronal integration of multiple signals may be determined by the biochemical characteristics of the AC that is expressed by the particular neuron. Because of the complexity and extensive involvement of AC in neuronal information processing, AC has been implicated in biological functions from synaptic plasticity and circadian rhythms (Chern, Y. (2000), Cell Signal 12:195-204).
It is well known that cAMP plays a major role in regulating cardiac function. Adult cardiac myocytes express multiple AC isoforms, with AC-5 as the dominant AC isoform. However, both the physiological significance of the existence of multiple isoforms and the role of AC-5 in the regulation of cAMP signal, in particular relative to that of AC-6, in intact heart have been very poorly understood.
Congestive heart failure (CHF) represents one of the major public health problems in most Western countries, but its pathophysiology remains largely unknown. Improved therapy could be offered to patients with heart failure if its molecular and genetic mechanisms were better defined.
Congestive heart failure (CHF) is defined as abnormal heart function resulting in inadequate cardiac output for metabolic needs (Braunwald, E. led), In: Heart Disease, W.B. Saunders, Philadelphia, page 426, 1988). Symptoms include breathlessness, fatigue, weakness, leg swelling, and exercise intolerance. On physical examination, patients with heart failure tend to have elevations in heart and respiratory rates, rales (an indication of fluid in the lungs), edema, jugular venous distension, and, in general, enlarged hearts. The most common cause of CHF is atherosclerosis, which causes blockages in the blood vessels (coronary arteries) that provide blood flow to the heart muscle. Ultimately such blockages may cause myocardial infarction (death of heart muscle) with subsequent decline in heart function and resultant heart failure. Other causes of CHF include valvular heart disease, hypertension, viral infections of the heart, alcohol, and diabetes. Some cases of heart failure occur without clear etiology and are called idiopathic.
CHF is also typically accompanied by alterations in one or more aspects of beta-adrenergic neurohumoral function; see, e.g., Bristow M R, et al., N Engl J Med 307:205-211, (1982); Bristow M R, et al., Circ Res 59:297-309, (1986); Ungerer M, et al., Circulation 87: 454-461, (1993); Feldman A M, et al., J Clin Invest 82:189-197, (1988); Bristow M R, et al., J Clin Invest 92: 2737-2745, (1993); Calderone A, et al., Circ Res 69:332-343, (1991); Marzo K P, et al., Circ Res 69:1546-1556, (1991); Liang C-S, et al., J Clin Invest 84: 1267-1275, (1989); Roth D A, et al., J Clin Invest 91: 939-949, (1993); Hadcock J R and Malbon C C: Proc NatI Acad Sci 85:5021-5025, (1988); Hadcock J R, et al., J Biol Chem 264: 19928-19933, (1989); Mahan, et al., Proc Natl Acad Sci USA 82:129-133, (1985); Hammond H K, et al., Circulation 85:269-280, (1992); Neumann J, et al., Lancet 2: 936-937, (1988); Urasawa K, et al., In: G Proteins: Signal Transduction and Disease, Academic Press, London. 44-85, (1992); Bohm M, Mol Cell Biochem, 147: 147-160, (1995); Eschenhage T, et al., Z Kardiol, 81 (Suppl 4): 33-40, (1992); and Yamamoto J, et al., J Mol Cell, 26: 617-626, (1994). See also the numerous additional references regarding various adenylylcyclase enzymes by, e.g., Fujita M et al., Circulation, 90: (No. 4 Part 2), (1994); Yoshimura M et al., Proc NatI Acad Sci USA, 89:6716-6720, (1992); Krupinski J et al., J Biol Chem, 267:24858-24862, (1992); Ishikawa Y et al., J Biol Chem, 267:13553-13557, (1992); Ishikawa Y et al., J. Clin Invest, 93:2224-2229, (1994); Katsushika S et al., Proc Natl Acad Sci USA, 89:8774-8778, (1992); Wallach J et al., FEBS Lett, 338:257-263, (1994); Watson P A et al., J Biol Chem, 269:28893-28898, (1994); Manolopoulos V G et al., Biochem Biophys Res Commun, 208:323-331, (1995); Yu H J et al., FEBS Lett, 374:89-94, (1995); and Chen Z et al., J Biol Chem, 270:27525-27530, (1995).
As a result of these studies and others, efforts to treat CHF have focused on the administration of pharmacological agents, such as catecholamines and other beta-adrenergic agonists, as means of stimulating beta-adrenergic responses in dysfunctional hearts. Such therapeutic approaches have been only partly successful. Furthermore, long-term exposure to catecholamines can be detrimental. In particular, the heart tends to become less responsive to beta-adrenergic stimulation, and such unresponsiveness is typically associated with high levels of catecholamines in plasma, a factor generally linked to a poor prognosis.
Present treatments for CHF include pharmacological therapies, coronary revascularization procedures (e.g. coronary artery bypass surgery and angioplasty), and heart transplantation. Pharmacological therapies have been directed toward increasing the force of contraction of the heart (by using inotropic agents such as digitalis and beta-adrenergic receptor agonists), reducing fluid accumulation in the lungs and elsewhere (by using diuretics), and reducing the work of the heart (by using agents that decrease systemic vascular resistance such as angiotensin converting enzyme inhibitors). Beta-adrenergic receptor antagonists have also been tested. While such pharmacological agents can improve symptoms, and potentially prolong life, the prognosis in most cases remains dismal.
Some patients with heart failure due to associated coronary artery disease can benefit, at least temporarily, by revascularization procedures such as coronary artery bypass surgery and angioplasty. Such procedures are of potential benefit when the heart muscle is not dead but may be dysfunctional because of inadequate blood flow. If normal coronary blood flow is restored, viable dysfunctional myocardium may contract more normally, and heart function may improve. However, revascularization rarely restores cardiac function to normal or near-normal levels in patients with CHF, even though mild improvements are sometimes noted.
Finally, heart transplantation can be a suitable option for patients who have no other confounding diseases and are relatively young, but this is an option for only a small number of patients with heart failure, and only at great expense. In summary, CHF has a very poor prognosis and responds poorly to current therapies.
Further complicating the physiological conditions associated with CHF, are various natural adaptations that tend to occur in patients with dysfunctional hearts. Although these natural responses can initially improve heart function, they ultimately result in problems that can exacerbate CHF, confound treatment, and have adverse effects on survival. There are three such adaptive responses commonly observed in CHF: (i) volume retention induced by changes in sodium reabsorption, which expands plasma volume and initially improves cardiac output; (ii) cardiac enlargement (from dilation and hypertrophy) which can increase stroke volume while maintaining relatively normal wall tension; and (iii) increased norepinephrine release from adrenergic nerve terminals impinging on the heart which, by interacting with cardiac .beta.-adrenergic receptors, tends to increase heart rate and force of contraction, thereby increasing cardiac output. However, each of these three natural adaptations tends ultimately to fail for various reasons. In particular, fluid retention tends to result in edema and retained fluid in the lungs that impairs breathing; heart enlargement can lead to deleterious left ventricular remodeling with subsequent severe dilation and increased wall tension, thus exacerbating CHF; and long-term exposure of the heart to norepinephrine tends to make the heart unresponsive to adrenergic stimulation and is linked with poor prognosis.
Controlled use of pharmacological agents, such as beta-adrenergic agonists; and other modulatory drugs, thus remains one of the major forms of treatment despite its shortfalls, including its potentially adverse effect on survival. Researchers who have analyzed and in some cases cloned DNA sequences encoding individual components involved in the beta-adrenergic receptor pathway have proposed using such components to identify new classes of drugs that might prove more useful in treating CHF. For example, Ishikawa et al. cloned DNA encoding two different isoforms of adenylyl cyclase (ACV and ACVI) that are known to be predominant in mammalian cardiac tissue, and proposed using the DNA and/or recombinant protein to identify new classes of drugs that might stimulate adrenergic pathways (See, e.g., American Cyanamid, WO 93/05061, Mar. 18, 1993, and EP 0 529 662, Mar. 3, 1993; and Ishikawa U.S. Pat. No. 5,334,521, issued Aug. 2, 1994). In other reports in which cloned components of the adrenergic stimulation pathway were investigated, the authors generated transgenic mice overexpressing certain components (including cardiac β2-adrenergic receptors, Gsα and G-protein receptor kinase inhibitors) and obtained some data suggesting that β-adrenergic stimulation may be enhanced in transgenic mice (see, e.g., Gaudin C, et al., J Clin Invest 95: 1676-1683, (1995); Koch W J, et al., Science 268: 1350-1353, (1995); and Bond R A, et al., Nature 364: 272-276, (1995)). None of these reports showed that cardiac function could be effectively restored in animals with heart failure, nor did they show that adrenergic responsiveness could be enhanced in large animal models that would be considered predictive of success in treating CHF in humans.
Indeed, reflecting on the observed difficulties associated with the clinical use of beta-adrenergic agonists (such as dopamine and dobutamine), a recent review concluded that beta-adrenergic stimulation appears to be harmful; and that, on the contrary, beta receptor “blockers” or antagonists may be more useful for improving morbidity and mortality rates (see, e.g., Baughman, K., Cardiology Clinics 13: 27-34, (1995)). While some agents may improve symptoms, the prognosis for patients receiving such pharmacological agents remains dismal.
As described herein, the expression of the type 5 AC isoform is limited to the heart and the striatum of the brain and is negligibly expressed in other tissues. The physiological significance of this cardiac and striatum-specific localization is unknown. To address this issue, and to help in identification of new therapeutic approaches for the treatment of cardiac and/or neurological impairment, the inventors of the present application have developed a transgenic mouse model lacking the expression of AC5. Furthermore, up until the time of the present invention, there was a lack of antibodies with the specificity needed to identify and quantify the type and amount of each isoform associated with a specific tissue. In particular, an AC5 monoclonal antibody with high specificity was not available up until the time of the present invention to distinctly identify and quantitate the presence of AC5 in specific tissue. The therapeutic and diagnostic utility of this AC5 specific monoclonal antibody is described herein.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.